In considering the genetic options for field resistance we had previously focused on the detoxification genes of two pests, Lucilia cuprina and Helicoverpa armigera. Over 100 genes of the Cytochrome P450 (Cyp), Carboxylesterase (CE) and Glutathione-s-transferase (Gst) gene superfamilies have been isolated. In 2005 we have gained a detailed understanding of when and where in these genes are expressed in H. armigera (Jeffrey Wee, Postgraduate Student). Comparable data will soon be available for L. cuprina. This type of analysis in combination with a comparative genomics approach is allowing us to prioritize the genes that are worthy of further analysis (outlined below).

When the original CESAR application was written, research on the detoxification genes of the model insect, D. melanogaster, was not included. This decision was made so that we could focus on the two pest insect systems. However, over the last three years the outcomes of the D. melanogaster research that we have conducted as a non-core activity, have strongly argued for the incorporation of this work under the SRC-CESAR umbrella. In brief, we have shown that field populations of D. melanogaster are resistant to almost all classes of chemicals used to control insect pests and that, in many cases, this resistance may be conferred by detoxification genes (Daborn et al., 2002). Building on Phillip Daborn’s work on the Cyp6g1 gene, we have been able to specifically overexpress eleven different D. melanogaster Cyp genes in metabolic tissues (midgut, fat body and malpighian tubules). None of these genes was previously known to have a role in resistance. We have shown that Cyp6g2, like Cyp6g1, is capable of conferring resistance to multiple insecticide classes. Cyp12d1 is capable of conferring resistance to DDT and the insect growth regulator dicyclanil. Six other D. melanogaster P450s tested are not capable of conferring resistance to any of the five insecticides tested (DDT, Diazinon, Nitenpyram, Lufenuron and Dicyclanil). We have also been investigating the capacity of Cyp6g1 orthologues from other Drosophila species to confer insecticide resistance (Tom Harrop, Honours Student). In this ongoing work, we have results indicating that Cyp6g1 from D. simulans can also confer DDT resistance. The Cyp6g1 orthologues from three different species have been cloned and overexpressed in D. melanogaster, but the resistance status of the overexpressing lines is yet to be determined. The long-term goal of the Cyp overexpression work is to generate the capacity to predict CYP function based on amino acid sequence data.

Many genes that encode detoxification enzymes are known to be induced by xenobiotic compounds. Jeffrey Wee and Lee Willoughby (Postgraduate Students) have examined the transcriptional induction of H. armigera and D. melanogaster Cyp, CE and Gst genes by xenobiotics in using microarrays. While compounds such as phenobarbital, piperonyl butoxide and caffeine induced a large fold increase in the transcription of many genes, none of the insecticides tested did so. Few genes were induced and even for these there was a low fold increase in mRNA levels compared with the controls. These data indicate that constitutive tissue specific overexpression, such as that generated by the Accord insertion in the Cyp6g1 gene of D. melanogaster, contributes more to resistance than induced responses. This finding has prompted us to invest further effort into the study of the regulation of detoxification genes. A preliminary bioinformatic analysis of the sequences 5’ to genes induced by phenobarbital, piperonyl butoxide and caffeine has not identified shared sequences that might transcription factors responsible for the induction. However, some candidate genes have been identified based on precedents from the mammalian literature.

The regulation of the Cyp6g1 gene has been under detailed investigation by Henry Chung (Postgraduate Student). Genomic regions responsible for Cyp6g1 expression in the key metabolic tissues (midgut and malpighian tubules) have been identified using 5’ deletions and GFP gene reporter constructs. A search for the transcription factors that bind to these DNA sequences, using yeast one hybrid analysis, has commenced. The impact of the Accord insertion on Cyp6g1 expression has also been addressed. Sequences within the Accord insertion have been shown to be responsible to a specific increase in Cyp6g1 expression in the midgut, fat body and malpighian tubules. Again, yeast one hybrid analysis is being used to find the transcription factors that bind to the Accord fragment in Cyp6g1. The methods established for the analysis of Cyp6g1 gene regulation will be applied to the study of Cyp12d1 in 2006.

The power to analyse the expression of the detoxification genes has been increased by our recent finding that some features of normal detoxification gene regulation can be studied in the highly manipulable S2 cultured cell system. In the 2006-8 triennium we will include research on the regulation of key detoxification genes a core activity.

It should be noted that the D. melanogaster research that we are adding to the core activity does not include the population genetics of resistance. Dr. Charles Robin has been appointed as a Lecturer at the University of Melbourne and has chosen to adopt this as a focus of his research. Dr. Robin has been appointed as an Associate of CESAR.